Methods for producing surfaces that resist non-specific protein binding and cell attachment

ABSTRACT

A method is disclosed herein for treating a polymeric surface to resist non-specific binding of biomolecules and attachment of cells. The method includes the steps of: imparting a charge to the polymeric surface to produce a charged surface; exposing the charged surface to a nitrogen-rich polymer to form a polymerized surface; exposing the polymerized surface to an oxidized polysaccharide to form an aldehyde surface; and exposing the aldehyde surface to a reducing agent. Advantageously, a method is provided which produces surfaces that resist non-specific protein binding and cell attachment and that avoids the use of photochemical reactions or prior art specially designed compounds.

This application claims priority of U.S. Provisional Patent ApplicationNo. 60/705,908, filed Aug. 5, 2005, which is incorporated by referenceherein.

FIELD OF THE INVENTION

The present invention relates to methods of treating plastic surfaceswhich resist non-specific protein binding or cell attachment, andsurfaces prepared by same.

BACKGROUND OF THE INVENTION

Bare plastic surfaces, such as polystyrene surfaces, typically do notresist non-specific protein binding or cell attachment. Surfacesmodified with a dense and stable layer of polymers such as polyethyleneglycol or hydrogels, such as dextran, are known to resist non-specificprotein binding and cell attachment. In the prior art, in order tocreate a dense and stable layer of protective polymers or hydrogels on aplastic surface, the plastic surface was typically treated with aphotochemical reaction to activate the surface or with prior artspecially designed chemicals that have a high affinity to the relevantsurface.

SUMMARY OF THE INVENTION

A method is disclosed herein for treating a polymeric surface to resistnon-specific binding of biomolecules and attachment of cells. The methodincludes the steps of: imparting a charge to the polymeric surface toproduce a charged surface; exposing the charged surface to anitrogen-rich polymer to form a polymerized surface; exposing thepolymerized surface to an oxidized polysaccharide to form an aldehydesurface; and exposing the aldehyde surface to a reducing agent.Advantageously, a method is provided which produces surfaces that resistnon-specific protein binding and cell attachment and that avoids the useof photochemical reactions or prior art specially designed compounds.

These and other features of the invention will be better understoodthrough a study of the following detailed description and accompanyingdrawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flowchart representing a method in accordance with thesubject invention.

FIG. 2 is a chart comparing the non-specific binding of Immunoglobin G(IgG) on two different surfaces: one surface is untreated and the othersurface was treated by the subject invention. The amount of IgG bound onthe surface was detected by the amount of IgG-HRP (horseradish peroxide)conjugate it could bind, and the amount of IgG-HRP conjugate wasquantified by the HRP catalyzed oxidation of TMB (3,3′,5,5′tetramethylbenzidine), which changes color upon oxidation.

DETAILED DESCRIPTION OF THE INVENTION

With reference to FIG. 1, a method 10 is depicted of treating apolymeric surface 12 to resist non-specific binding of biomolecules andattachment of cells.

In an initial step 30, a charge is provided to the polymeric surface 12of a vessel or receptacle to produce a charged surface 14. The vesselmay be of any known configuration, such as a test tube, vial, flask,etc. Preferably, the polymeric surface 12 is the surface of a multiwellplate. More preferably, the polymeric surface 12 is a surface of a wellof a multiwell plate. It is further preferred that the multiwell plateconform to conventional multiwell plate standards (e.g., the Standardsof the Society of Biomolecular Screening) so as to be usable in drugassay handling equipment (e.g., high throughput screening (HTS)equipment).

The term “polymeric surface” as used herein refers to any suitable suchpolymeric surface known to those skilled in the art. Suitable examplesof polymeric surfaces include those obtained from polymerichydrocarbons. As used herein, the term “polymeric hydrocarbon” isintended to refer to those polymers and copolymers obtained fromrepeating monomer units which are composed of carbon and hydrogen. Thepolymeric hydrocarbons may be saturated or unsaturated, and substitutedor unsubstituted. Substituents may include atoms other than hydrogen andcarbon, as long as they are present in an amount that does not detractfrom the substantially hydrocarbon nature of the polymer. Suchsubstituents include acetal, halo, hydroxy, cyano, alkoxy, amino, amido,carbamoyl, and carbamido groups. Typical examples of a polymerichydrocarbon surface include those made from substituted andunsubstituted polyethylene, polypropylene, polystyrene, ABS, PVC,polytetrafluoroethylene, polyvinylidene, and mixtures thereof. In apreferred embodiment, the polymeric hydrocarbon surface is polystyrene.

The term “polymeric surface” is also intended to include surfacesobtained from those polymers containing one or more heteroatoms such asoxygen, nitrogen, or sulfur, in addition to carbon and hydrogen. Typicalexamples of such polymeric surfaces include surfaces obtained fromsubstituted and unsubstituted polyethers, polyesters, polyamides,polyamines, polyimines, polyurethanes, polyrureas, polyacetals,polycarbonates, polyacrylates, polysulfides, polysulfones, andpolysulfides.

Also contemplated as being within the scope of the present invention aresurfaces obtained from polymers with backbones composed significantly ofheteroatoms, such as silicones.

Any known technique can be used to impart the charge to the polymericsurface 12 to produce the charged surface 14. Preferably, plasmatreatment or corona discharge treatment may be utilized. With thisprocess, a charge is imparted to the polymeric surface 12 by disposingthe polymeric surface 12 into a substantially gas-free chamber,introducing a gas into the chamber, and exciting the gas. As a result,plasma is formed and applied to the polymeric surface 12 to produce thecharged surface 14. A high-frequency generator may be used to ionize thegas into a plasma. In addition, the plasma may be generated usingconventional plasma conditions such AC or DC power levels up to about200 watts, radiofrequency (RF) excitation of about 0.1 to about 50megahertz, for a durations of about 0.1 to about 30 minutes, with a gaspressure of about 0.1 to about 3.0 Torr. A conventional plasma chambermay be used, although it is preferred that the chamber be evacuatedduring use.

Although an RF excited plasma is preferred, any other method ofgenerating a gas plasma may be used, for example a glow discharge or acorona discharge. For example, microwave frequencies may be employedinstead of, or in addition to, RF excitation.

Gases typically used with plasma treatment and introduced into theplasma chamber include Ar, He, Ne, He, He/H₂, O₂, N₂, NH₃, and CF₄. Inone embodiment of the invention, the charged surface 14 may benegatively charged. A negatively charged surface is specificallydesignated with reference numeral 14(a) in FIG. 1. Preferably, oxygengas is used in the plasma treatment process to produce the negativelycharged surface 14(a).

Alternatively, in another embodiment, the charged surface 14 may bepositively charged. A positively charged surface is specificallydesignated with reference numeral 14(b) in FIG. 1. Preferably, ammoniagas is used in the plasma treatment process to produce the positivelycharged surface 14(b). Specifically, subjecting the polymeric surface 12to ammonia gas plasma treatment creates a number of nitrogen containing,positively charged functional groups on the surface, providing thepositively charged surface 14(b).

In a next step 32 of the method 10, the charged surface 14 is exposed toa nitrogen-rich polymer to form a polymerized surface 16. The negativelycharged surface 14(a) may be exposed to the nitrogen-rich polymerwithout any intervening steps. However, before the positively chargedsurface 14(b) may be exposed to the nitrogen-rich polymer, thepositively charged surface 14(b) is preferably first exposed to one ormore suitable linkers. A variety of linkers, commonly referred to as“cross-linkers” may be used. Suitable linkers include: dialdehydes,diesters, diimidoesters, NHS-esters, hydrazides, carbodiimides, and arylazides. Also contemplated as being within the scope of the invention areheterobifunctional linkers, i.e. those which have different functionalgroups on each end. For example, a suitable heterobifunctional linkerwould be one having an ester on one end and an aldehyde on the otherend. In a preferred embodiment, the linker is a dialdehyde having thestructure:

wherein R¹ is a C₂ to C₃₀ alkylenyl. In a more preferred embodiment, thedialdehyde is glutaraldehyde.

Preferably, the positively-charged surface 14(b) is exposed to asolution of the linkers. Any suitable solvent or suitable mixture ofsolvents known to those skilled in the art may be used with the linkers.Suitable solvents include water, buffers, methanol, ethanol,isopropanol, and dimethylsulfoxide (DMSO).

Once readied, the charged surface 14 is exposed to a nitrogen-richpolymer to form the polymerized surface 16. The term “nitrogen-rich” isintended to refer to polymers bearing pendant amino groups such asN(R²)₂ and ═NR², wherein each R² is independently H or C₁ to C₁₀ alkyl.As used herein, the term “alkyl” intended to refer to branched andstraight-chained saturated aliphatic hydrocarbon radicals having theindicated number of carbon atoms. Alkyl groups may be unsubstituted, orsubstituted. Suitable substituents include C₁₋₅ alkyl, amino, amido,cyano, carbamoyl, phenyl, heteroaryl, halogen, C₁₋₅ alkoxy, C₁₋₅alkyl-C(O)H, CO₂H, and CO₂-C₁₋₅ alkyl. The term “alkylenyl” is intendedto encompass diradical variations of alkyl groups.

Preferably, the nitrogen-rich polymer is a polyalkylenimine such aspolyethylenimine. Another class of nitrogen-rich polymers suitable forthe present invention is polymeric amino acids. The term “polymericamino acid” is intended to refer to a string of repeating amino acids.Accordingly, any suitable peptide may be used as a nitrogen-richpolymer. The string of amino acids may contain a string of identicalamino acids or a string of different amino acids, and in either case maybe natural or man-made. Nitrogen-rich polymers based on amino acids suchas lysine and arginine possess sufficient nitrogen character so as to begood examples of suitable nitrogen-rich polymers. A synthetic polymericamino acid particularly useful in the present invention as a polymericamino acid is poly-lysine. In a more preferred embodiment, the syntheticpolymeric amino acid is poly-d-lysine.

Typically, the charged surface 14 will be exposed to a solution of thenitrogen-rich polymer, forming the polymerized surface 16. Any suitablesolvent or suitable mixture of solvents known to those skilled in theart may be used. Suitable solvents include water, buffers, methanol,ethanol, isopropanol, and dimethylsulfoxide (DMSO).

In the next step 34, the polymerized surface 16 is exposed to analdehyde-bearing polymer, thereby providing aldehyde surface 18. Anypolymer bearing pendant hydroxyalkyl groups can serve as thealdehyde-bearing polymer. Preferably, the alcohols on such a polymer areoxidized to aldehydes, with the aldehydes being receptive to couplingwith both the nitrogens of the polymerized surface 16 and the nitrogensof an outer layer discussed below. However, because the aldehyde surface18 must be biologically benign, it is preferred that the alcohol-bearingpolymer not be toxic to biological or cell cultures. Preferably, thealdehyde-bearing polymer is an oxidized polysaccharide in which thependant alcohol groups have been converted to aldehyde groups. Suitableoxidized polysaccharides include oxidized polysaccharides such asoxidized amylose, oxidized amylopectin, oxidized cellulose, oxidizedchitin, oxidized guaran, oxidized glucomannan, and oxidized dextran.Among these, oxidized dextran is particularly preferred. In a preferredmethod, the polysaccharides are oxidized by adding sodium m-periodate(NaIO₄) to the polysaccharide solution, with the resulting solutionbeing incubated at room temperature in the dark for 4 hours, followed byremoval of the sodium m-periodate (e.g., by dialysis).

Typically, the polymerized surface 16 will be exposed to a solution ofthe aldehyde-bearing polymer to form the aldehyde surface 18. Anysuitable solvent or suitable mixture of solvents known to those skilledin the art may be used. Suitable solvents include water, buffers,methanol, ethanol and isopropanol.

The aldehyde surface 18 is further treated, as shown in step 36, whichmay involve one step or two sub-steps, in forming a stabilized surface20.

In one embodiment, the polymerized surface 18 may be exposed to areducing agent, thereby producing the stabilized surface 20,specifically designated for this embodiment as stabilized surface 20(a)in FIG. 1. Preferably, the reducing agent is a boron-based reducingagent such as NaBH₄ or NaCNBH₃.

Alternatively, in another embodiment, the polymerized surface 18 isfirst exposed to an amine-terminated polymer. Preferably theamine-terminated polymer is an amine-terminated hydrocarbyl polymer oran amine-terminated polyether. The term “hydrocarbyl polymer” isintended to be synonymous with the term “polymeric hydrocarbon” asdiscussed hereinabove. In a more preferred embodiment, theamine-terminated polyether is amine-terminated polyethylene glycol.Typically, the amine-terminated polymer will be dissolved in suitablesolvent when exposed to polymerized surface 18. Any suitable solvent orsuitable mixture of solvents known to those skilled in the art may beused. Suitable solvents include water, buffers, methanol, ethanol andisopropanol.

Reaction of the aldehyde surface 18 and the amine groups of theamine-terminated polymer forms a reversible Schiff base linkage whichcan then be stabilized with a suitable reducing agent, thereby producingstabilized surface 20, specifically designated for this embodiment asstabilized surface 20(b) in FIG. 1. The suitable reducing agent is asdescribed above with respect to the stabilized surface 20(a).

EXAMPLES Example A

A polystyrene surface is exposed to oxygen gas plasma treatment,creating a negatively charged surface.

The negatively charged surface is exposed to a solution of 1%polyethylenimine for 2 hours. The polyethylenimine coated surface isexposed to a solution of 10 mg/mL oxidized dextran for two hours. Thedextran coated surface is exposed to a solution of amine-terminatepolyethylene glycol for 1 hour. The polyethylene glycol surface isexposed to a solution of 1 mg/mL sodium borohydride for 1 hour.

Example B

A polystyrene surface is exposed to ammonia gas to create a positivelycharged surface. The positively charged surface is exposed to a solutionof 10% glutaraldehyde for 1 hour. The glutaraldehyde activated surfaceis exposed to a solution of 1% polyethylenimine for 2 hours. Thepolyethylenimine coated surface is exposed to a solution of 10 mg/mLoxidized dextran for 2 hours. The dextran coated surface is exposed to asolution of 1 mg/mL amine-terminated polyethylene glycol for 1 hour. Thepolyethylene glycol coated surface is exposed to a solution of 1 mg/mLsodium borohydride for 1 hour.

As will be appreciated by those skilled in the art, the subjectinvention provides polymeric surfaces which will resist non-specificbinding of biomolecules and attachment of cells. The stabilized surface20 provides such resistance. With reference to FIG. 2, data is presentedrelating to the non-specific binding of IgG on two different surfaces:surfaces not treated by the method of the subject invention and surfaceswhich have been treated by the subject invention. In this demonstration,a 96-well polystyrene plate was treated using the method of Example A.Another 96-well polystyrene plate was not treated and was used as areference. The surfaces in the wells of both of the plates were broughtinto contact with 5 μg/mL of anti-mouse IgG for 2 hours followed bywashing with PBS (phosphate buffered saline). Then the surfaces werebrought into contact with mouse IgG-HRP (horseradish peroxide) conjugate(concentration ranges from 0.01 μg/mL to 0.33 μg/mL) for 1 hour followedby washing with PBS. Thereafter, the surfaces were brought into contactwith TMB (3,3′,5,5′ tetramethylbenzidne) solution for 8 minutes followedby adding 2N HCl to stop the reaction. The amount of anti-mouse IgG andthe associated mouse IgG-HRP conjugate bound on the surfaces wasquantified by the intensity of the color (detected at 450 nm) producedby the oxidized TMB. As can been seen in FIG. 2, negligible amounts ofImmunoglobin G were absorbed by the treated surfaces.

Experiments have been conducted relating to the attachment of varioustypes of adherent cells on two different surfaces: surfaces not treatedby the method of the subject invention and surfaces which have beentreated by the subject invention. In the following describedexperiments, a 6-well polystyrene plate was treated using the method ofExample A. Another 6-well polystyrene plate was untreated and used as areference.

In a first experiment, HT-1080 (human fibrosarcoma cell line) cells werecultured on both untreated and treated surfaces of 6-well plates underthe same culture condition (incubation at 37° C. in growth media). Cellattachment and spreading on the surfaces were analyzed and microscopicimages were taken following several days of cell culture. The HT-1080cells attached to the untreated surface and spread on the surface asexpected. However, the HT-1080 cells remained un-attached to the treatedsurface and formed cell aggregates floating in the media. The treatedsurface remained free of cells after removing the media, demonstratingthe ability of the treated surface for resisting HT-1080 cellattachment.

In a second experiment, mouse embryo fibroblasts (NIH/3T3) were culturedon both untreated and treated surfaces of 6-well plates under the sameculture condition (incubation at 37° C. in growth media). Cellattachment and spreading on the surfaces were analyzed and microscopicimages were taken following several days of cell culture. Thefibroblasts attached to the untreated surface and formed a monolayer onthe surface as expected. However, the fibroblasts remained un-attachedto the treated surface and formed cell aggregates floating in the media.The treated surface remained free of cells after removing the media,demonstrating the ability of the treated surface for resistingfibroblast attachment.

In a third experiment, canine chondrocytes were cultured on bothuntreated and treated surfaces of 6-well plates under the same culturecondition (incubation at 37° C. in growth media). Cell attachment andspreading on the surfaces were analyzed and microscopic images weretaken following several days of cell culture. The chondrocytes attachedto the untreated surface and spread on the surface as expected. However,the chondrocytes remained un-attached to the treated surface and formedcell aggregates floating in the media. The treated surface remained freeof cells after removing the media, demonstrating the ability of thetreated surface for resisting chondrocyte attachment.

Experiments have been conducted relating to the formation of embryoidbodies from embryonic stem cells. The formation of embryoid bodies wassuccessfully achieved using the 6-well polystyrene plates treated by themethod of the subject invention. Untreated 6-well polystyrene plateswere used as controls and embryoid bodies did not form due to theattachment of embryonic stem cells to the untreated surfaces during thelong incubation time (up to 7 days). With the treated surfaces,attachment of the embryonic stem cells was generally avoided, and theembryonic stem cells remained in suspension during incubation. As such,without attachment, the embryonic stem cells generally avoidedattachment-mediated differentiation, thereby permitting later enhancedembryoid body formation.

The subject invention may have applicability in various contexts. By wayof non-limiting examples, the subject invention can be used to preparepolymeric surfaces to obtain the following advantages: maintaining cellsin solution in suspended, unattached states; preventing stem cells fromattachment-mediated differentiation; permitting enhanced formation ofembryoid bodies from embryonic stem cells; preventinganchorage-dependent cells from dividing; reducing binding of serumproteins; and, enhancing signal-to-noise ratios in homogenous assays,such as Scintillation Proximity Assays.

1. A method for treating a polymeric surface to resist non-specificbinding of biomolecules and attachment of cells, the method comprisingthe steps of: a) imparting a charge to the polymeric surface to producea charged surface; b) exposing the charged surface to a nitrogen-richpolymer to form a polymerized surface; c) exposing the polymerizedsurface to an aldehyde-bearing polymer to form an aldehyde surface; andd) exposing the aldehyde surface to a reducing agent.
 2. The method ofclaim 1, wherein the step of exposing the aldehyde surface to thereducing agent further includes exposing the aldehyde surface to anamine-terminated polymer selected from the group consisting ofamine-terminated hydrocarbyl polymers and amine-terminated polyethers,and then exposing the surface to the reducing agent.
 3. The method ofclaim 2, wherein the amine-terminated polyether is amine-terminatedpolyethylene glycol.
 4. The method of claim 1, wherein the step ofimparting the charge to the polymeric surface includes disposing thepolymeric surface into a substantially gas-free chamber, introducing agas into the chamber, and exciting the gas to produce the chargedsurface.
 5. The method of claim 4, wherein the exciting of the gasincludes subjecting the gas to radiofrequency excitation.
 6. The methodof claim 4, wherein the gas is oxygen gas.
 7. The method of claim 4,wherein the gas is ammonia gas.
 8. The method of claim 7, furthercomprising the step of exposing the charged surface to a linker prior tostep b).
 9. The method of claim 8, wherein the linker is selected fromthe group consisting of: dialdehydes, diesters, diimidoesters,NHS-esters, hydrazides, carbodiimides, and aryl azides.
 10. The methodof claim 8, wherein the linker is glutaric aldehyde.
 11. The method ofclaim 1, wherein the nitrogen-rich polymer is selected from the groupconsisting of: a polyalkylenimine and a polymeric amino acid.
 12. Themethod of claim 11, wherein the nitrogen-rich polymer is selected fromthe group consisting of: polyethylenimine and poly-lysine.
 13. Themethod of claim 1, wherein the aldehyde-bearing polymer is an oxidizedpolysaccharide.
 14. The method of claim 13, wherein the oxidizedpolysaccharide is selected from the group consisting of: oxidizedamylose, oxidized amylopectin, oxidized cellulose, oxidized chitin,oxidized guaran, oxidized glucomannan, and oxidized dextran.
 15. Themethod of claim 1, wherein the polymeric surface is a polystyrenesurface.
 16. The method of claim 1, wherein the polymeric surface is asurface of a multiwell plate.
 17. The method of claim 1, wherein thereducing agent is sodium borohydride.
 18. A surface treated by themethod of claim
 1. 19. The surface of claim 17, wherein the surface isthe surface of a multiwell plate.